Biomaterials based on hyaluronic acid for the anti-angiogenic therapy in the treatment of tumors

ABSTRACT

The use in the medical-surgical field of biomaterials based on hyaluronic acid derivatives, optionally in association with natural, synthetic or semisynthetic biopolymers, for suppressing the angiogenic process associated with tumour proliferation (in primary and secondary tumours) is disclosed.

SUBJECT OF THE INVENTION

The present invention relates to the use in the medical-surgical fieldof biomaterials based on hyaluronic acid derivatives, optionally inassociation with natural, synthetic or semisynthetic biopolymers, forsuppressing the angiogenic process associated with tumour proliferation(in primary and secondary tumours).

BACKGROUND OF THE INVENTION

The induction and development of angiogenesis is a pre-requisite for thedevelopment of a primary tumour, and for any subsequent metastases.

Angiogenesis is a dynamic process closely linked with the proliferationof cancer cells, because it is the latter that are chiefly responsiblefor the production and release of angiogenic factors, such as cytokinesand other trophic factors. An increase in the vascularisation of aprimary tumour can cause an increase in the number of cancer cells thatenter into the circulation and give rise to new metastases.

Recent studies have demonstrated that an increase in the density ofmiorovessels in an area affected by neoplasia indicates new tumourgrowth.

It is therefore clinically important to suppress angiogenesis to inhibitits development, if possible. Indeed, by associating anti-angiogenictherapy with “classic” anticancer therapy with drugs and/or radiation,with or without surgical removal of the tumour, it is possible to haltthe proliferation of cancer cells, thus preventing the invasion offurther tissues by said cells, and the consequent development of newmetastases (Skobe H. et al., Nature Medicine, 1222-1227 (1997)).

In histological assessment of the onset of the angiogenic processassociated with a cancerous growth, it is important to look for markersof the tumour's vascular system, for example with antibodies thatdifferentiate the endothelial cells from the cancerous ones. Forexample, the anti-CD3 antibody is specific for marking the endothelialcells and therefore enables their identification in the angiogenicprocess associated with the development of new metastases. Its use hasproved essential in assessing the level of microvessel developmentassociated with neoplasia. Indeed, thanks to antibody marking, it ispossible to visualise and count the number of interconnections of thevessels within the cancerous tissue to understand and quantify theangiogenic process, relating it to any new developments in the neoplasia(thereby deciding if/how much/how to associate a therapy that modulatesor inhibits angiogenesis with an established/classic anticancer therapy.

One such therapy consists in administering drugs that act by blockingthe receptors of the trophic factors (PGDF, bFGF, VEGF) that are alsoangiogenic factors.

Preclinical results ‘in vivo’ have shown that said drugs prove importantin inhibiting tumour growth but they do not determine regression of thetumour itself: on the strength of these major experimental data, thedrugs have been introduced in numerous clinical trials.

However, an anti-angiogenic clinical therapy that provides for agenerally oral pharmacological administration in chronic form may havemany toxic side effects, because angiogenesis is not only associatedwith pathological disorders but also physiological processes such astissue reproduction and repair (“Cancer: Principle Practice of Oncology”V. De Vita, S. Hellmann and S. Rosenberg, 6^(th) Edition).

It is therefore of strategic importance to associate classic anticancertherapy with an anti-angiogenic therapy “in situ”, and this is thesubject of the present invention.

Hyaluronic acid is one of the chief components of the extracellularmatrix of the connective tissue, and there are numerous scientificpublications concerning its role in various processes, bothphysiological and pathological, such as the formation of granulationtissue, chemotaxis in the inflammatory process, cell differentiation forvarious cell types. Other studies concern its role within the family of“substrate adhesion molecules”.

Hyaluronic acid has been used for the above indications:

-   -   as a differentiating agent in therapy for acute myeloid        leukaemia (Charrad R. S. et al., Nature Medicine 5, 669-676        (1999));    -   as a vehicle for drugs such as steroids or NSAIDs, antibiotics        and anti-neoplastic agents, because of the abundant expression        of its receptor (CD44) in cancer cells; (Freemantle, C. et al.,        Int. J. Tiss. Reac. XVIII (4) 157-166 (1995); Coradini, D. et        al., Int. J. Cancer 5, 411-416 (1999));    -   in preclinical studies on the inhibition of lung metastasis,        because of its capacity for inhibiting the adhesion of cancer        cells to the vascular endothelium (Karasaza K. et al., Clinical        & Experimental Metastasis 15, 83-93 (1997));    -   as a means of controlling adhesion to the substrate with        subsequent proliferation of cells (possibly also cancer cells)        permanently “in situ” after surgical removal of tissues        (including tumours) (U.S. Pat. No. 5,627,162).

Experimental observations “in vivo” have, however, revealed thathyaluronic acid may have a chemotaxic activity on cancer cells withinthe granulation tissue that forms after removal of cutaneous metastasisof melanoma (Salmon-Ehr, V. et al., Ann. Dermatol. Venereol, 123,194-195 (1996)). Moreover, numerous pre-clinical studies havedemonstrated that hyaluronic acid enhances cancer cell migration,thereby favouring metastasis, as it is known that the degradationproducts of hyaluronic acid, that is, oligosaccharides constituted by 10and 20 oligomers, are strong inducers of the anigiogenic process (Hayenet al., J. Cell. Sci. 112, 2241-2251 (1999); Slevin, M. et al., Lab.Invest. 78 (8), 987-1003 (1998)).

Moreover, biomaterials based on hyaluronic acid and/or the derivativesthereof have never been used as an anti-angiogenic therapy, neither haveany other biodegradable and/or non-biodegradable biopolymers ever beenused in anticancer therapies.

Absolutely innovative, therefore, is the use of biomaterials based onhyaluronic acid derivatives such as Hyaff® (EP 0 216 453 B1) or ACPs (EP0 341 745 B1) in the form of non-woven felts for instance (EP0 618 817B1) or as three-dimensional structures (WO 99/61080), possibly inassociation with various biomaterials (e.g. natural ones such ascollagen, cellulose, polysaccharides, chitin, chitosan, pectin, agar,gellan and alginic acid, synthetic ones such as polylactic acid (PLA),polyglycolic acid (PGA), polyurethanes and polysulphonic resins, orsemisynthetic ones such as collagen cross-linked with aldehyde, diamineand gellan) as a therapy to suppress and/or inhibit the angiogenicprocess that enhances and determines tumour metastasis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to biomaterials based on hyaluronic acidderivatives made into non-woven felts (as the preferred form ofbiomaterial), optionally in association with natural, synthetic orsemisynthetic biopolymers, for use in the medical-surgical field as anew anti-angiogenic therapy (“in situ”), optionally associated withclassic pharmacological anticancer therapies and/or radiotherapy, tomodulate indirectly the proliferation of tumours, thus blocking theformation of local relapses and, therefore, any new metastases.

In order to study, characterise and then assess “in vivo” the effect ofthe biomaterial of the present invention in the angiogenic process thatsupports the development of skin carcinomas (considered to be aclarifying example), the Applicant has developed a new model oftumour/stromal cell support interaction, described as follows:

-   -   1) two cell lines of human keratinocytes transfected with the        ras-oncogene: HACaT II-4, malignant variant and A5, benign        variant;    -   2) said cells are transferred onto a collagen gel mounted into        teflon rings covered by a silicone chamber, known as the Fusenig        silicone chamber (FSC);    -   3) said FSC is then placed over the muscle fascia of the backs        of nude mice, in the presence or absence of an immediately        underlying layer of biomaterial based on Hyaff® 11 (total benzyl        ester of hyaluronic acid) made in the form of a non-woven felt;    -   4) four to six weeks later, two different types of granulation        tissue will have formed underneath the cancerous epithelium;    -   5) the development of the epithelial tumour and of the        underlying granulation tissue is assessed, over time, both by        classic histological analyses (haematoxylin/eosin) and by        immunohistochemical techniques using the anti-CD31 antibody, to        visualise the presence of vascular epithelium and therefore        determine the development of the angiogenic process;    -   6) the levels of cellular proliferation are examined using        immunohistochemical techniques associated with the introduction        of BrdU into the DNA of proliferating cells, both within the        granulation tissue underneath the epithelium and in the        cancerous epithelium itself. Marking with the anti-integrin α6        antibody was also assessed to study the level of cellular        proliferation within the cancerous epithelium.

The results of the experiment were as follows:

HACaT A5 line:

After 4-6 weeks, the cancerous epithelium in the control FSC (i.e.without any biomaterial placed under the epithelium), was well developedand multilayered, while the layer of granulation tissue underneath hadcompletely replaced the layer of collagen that separated the epitheliumfrom the underlying tissue (FIG. 1).

Conversely, four weeks later, the cancerous epithelium in the FSC placedover the Hyaff®-based biomaterial in the form of a non-woven felt isless developed than the relative control, and the layer of collagen thatseparates it from the nascent granulation tissue underneath is stillthick and not infiltrated by cells and/or vessels (FIG. 1).

After six weeks, the quantity of collagen is still abundant, with justan initial layer of granulation tissue that begins to form over thebiomaterial (FIG. 1).

HACaT II 4 Line:

After four to six weeks, in the control FSC, the cancer cells haveconstituted a thick epithelium that penetrates into the thickness of thenew granulation tissue underneath, that has already completely replacedthe layer of collagen that separated it from the epithelium (FIG. 2).

Four weeks later, in the FSC placed over the Hyaff®-based biomaterial,the cancerous epithelium is thin but easily distinguishable from thegranulation tissue forming over the biomaterial, separated from thistissue by the collagen gel that is still present and not yet absorbed(FIG. 2).

Six weeks later, the tumour mass and the granulation tissue haveestablished close contact, but there has been no actual infiltration oftumour cells into the granulation tissue, unlike the control, where thetumour cells have completely invaded the new, underlying granulationtissue (FIG. 2).

Using immunohistochemical techniques linked with the specific marking ofparticular nucleotides such as BrdU, at the 1^(st) and 2^(nd) weeks,good cell proliferation is evident within the nascent granulation tissuein the control and in the Hyaff®-based biomaterial, while at 4, andespecially at 6, weeks after transplant, the cell growth rate dropsdrastically in the granulation tissue underneath the cancerousepithelium, which conversely maintains in both samples a good level ofcellular proliferation (FIG. 3).

The growth of cancerous epithelium can also be visualised with aspecific antibody against the protein integrin α6. Said molecule is,indeed, a component of the hemidesmosomes and its expression is normallyonly associated with the proliferative area of the epithelial layers.

FIG. 3 a shows that antibody marking for the integrin protein α6 isnotably present throughout the cancerous epithelium both in the controlFCS and in the FCS with the Hyaff®-based biomaterial, even thoughexpression of the test protein appears less extensive throughout thethickness of the cancerous epithelium in the latter sample.

Specific marking for the vascular epithelium with the anti-CD31 antibodyreveals, furthermore, that in the controls, after four weeks, theangiogenic process is well established as the vessels in the underlyinggranulation tissue already reach the cancerous epithelium and after 6weeks they invade it, thus favouring metastasis (FIG. 4).

In the case of the FSC with the Hyaff®-based biomaterial, after fourweeks there is still no close contact between granulation tissue andcancerous epithelium. This will occur only after six weeks, even thoughthere is no invasion of the epithelium by the underlying microvessels,that remain relegated to the granulation tissue (FIG. 4).

The angiogenic process seems to be at a standstill, no longer enhancingtumour development. Vascularisation is limited to the area covered bythe Hyaff®-based biomaterial, so the tumour cells do not invade thegranulation tissue that has formed within the biomaterial.

In conclusion, the Hyaff®-based biomaterial proved able tomodulate/inhibit the angiogenic process related to vascularisation ofthe cancerous epithelium. It therefore proves to be particularlyadvantageous to use the biomaterials based on hyaluronic acidderivatives in the oncological field, where it is important to modulatethe angiogenic process and therefore, indirectly, the proliferation ofcancer cells in primary and secondary tumours.

According to the invention, the biomaterials that can be useful in theoncological field as a new anti-angiogenic therapy “in situ” may be, forexample, in the form of non-woven felts, sponges, films, membranes,microspheres or in other three-dimensional forms in cases where it isnecessary to fill the cavities that are liable to form after surgicalremoval of a tumour.

The anti-angiogenic action of the biomaterial can, moreover, besupported by supplementing the biomaterial with NSAIDs, steroids,hormones, antibiotics and especially anti-cancer drugs such asfluorouracil, methotrexate, cis-platinum, carboplatin, oxaliplatin,ethopoxide, cyclophosphamide, vincristine, doxorubicin.

The invention being thus described, it is clear that these methods canbe modified in various ways. Said modifications are not to be consideredas divergences from the spirit and purposes of the invention and anymodification that would appear evident to an expert in the field comeswithin the scope of the following claims.

1. Use of benzyl ester of hyaluronic acid or a cross-linked derivativeof hyaluronic acid wherein the carboxy groups of hyaluronic acid arecross-linked to the hydroxyl group of the same or different hyaluronicacid molecule, for the preparation of a biomaterial suitable forantiangiogenic therapy to treat primary and secondary tumours.
 2. Theuse according to claim 1 wherein hyaluronic acid is in association withother natural, synthetic and/or semisynthetic biopolymers.
 3. The useaccording to claim 2, wherein the natural biopolymer is selected fromthe group consisting of collagen, cellulose, polysaccharides, chitin,chitosan, pectins, agar, gellan and alginic acid.
 4. The use accordingto claim 2, wherein the synthetic biopolymer is selected from the groupconsisting of polylactic acid (PLA), polyglycolic acid (PGA),polyurethanes and polysulphonic resins.
 5. The use according to claim 2,wherein the semisynthetic biopolymer is selected from the groupconsisting of collagen cross-linked with aldehydes, diamine and gellan.6. The use according to claim 1 wherein the biomaterial is associatedwith pharmacologically active substances.
 7. The use according to claim6, wherein the pharmacologically active substance is selected from thegroup consisting of fluorouracil, methotrexate, cis-platinum,carboplatin, oxaliplatin, ethopoxide, cyclophosphamide, vincristine,doxorubicin.
 8. The use according to any one of claims 1-7 wherein thebiomaterial is in the form of a non-woven felt, sponge, microsphere,film or membrane and/or other three-dimensional structures.
 9. The useaccording to claim 8, for the treatment and care of primary andsecondary tumours when the tumour has been surgically removed and thecavity that is thus formed requires filling.
 10. A method for thetreatment and care of primary and secondary tumors which comprisesfilling a cavity resulting from the surgical removal of a tumor with abenzyl ester of hyaluronic acid or a cross-linked derivative ofhyaluronic acid wherein the carboxy groups of hyaluronic acid arecross-linked to the hydroxyl group of the same or different hyaluronicacid molecule.
 11. The method according to claim 10 wherein hyaluronicacid is in association with other natural, synthetic and/orsemisynthetic biopolymers.
 12. The method according to claim 2, whereinthe natural biopolymer is selected from the group consisting ofcollagen, cellulose, polysaccharides, chitin, chitosan, pectins, agar,gellan and alginic acid.
 13. The method according to claim 2, whereinthe synthetic biopolymer is selected from the group consisting ofpolylactic acid (PLA), polyglycolic acid (PGA), polyurethanes andpolysulphonic resins.
 14. The method according to claim 2, wherein thesemisynthetic biopolymer is selected from the group consisting ofcollagen cross-linked with aldehydes, diamine and gellan.
 15. The methodaccording to claim 10 wherein the biomaterial is associated with atleast one pharmacologically active substance.
 16. The method accordingto claim 15, wherein the pharmacologically active substance is selectedfrom the group consisting of fluorouracil, methotrexate, cis-platinum,carboplatin, oxaliplatin, ethopoxide, cyclophosphamide, vincristine; anddoxorubicin.
 17. The method according to any one of claims 10-16 whereinthe biomaterial is in the form of a non-woven felt, sponge, microsphere,film or membrane and/or other three-dimensional structure.